The molecular mechanism(s) which govern tissue and developmental specificity of human beta-globin gene regulation has been studied in K562 cells, a human erythroleukemia cell line that expresses high levels of embryonic and fetal globins beta-"like" globin. Structural analysis, including gel mobility shift and DNAse I hypersensitive footprinting techniques, revealed that the 5' upstream regulatory region of the beta-globin gene contains three structural motifs (-530, -302, -289) that are bound by two putative trans-acting factors, termed beta-protein 1 and beta-protein 2 (BP1, BP2). In situ mutagenesis of each structural motif introduced into a reporter vector containing chloramphenicol acetyltransferase (pCAT) followed by transient transfection into hemin-induced K562 cells yielded increased levels of CAT activity as much as 5.5-fold (ranging from 49% to 68% CAT induction, p<0.001) compared to wild type (wt) & RSVCAT and therefore further confirmed these distal promoter sequences as silencers of beta-globin expression. Mutagenesis of all three cis-acting elements however, resulted in CAT activity lower than wt beta-CAT (5% versus 13%, p less than 0.05). Sequence analysis revealed the beta -globin motifs for BP1 and BP2 have overlapping binding sites with the chromosomal high mobility group protein (HMG1+2), a non sequence-specific DNA-binding and - bending factor shown here by circular permutation analysis to extrinsically bend the beta-globin gene. In theory, the removal of BP1 and BP2 binding sites also disturbed the binding of HMG1+2 and thus impeded DNA-protein and/or protein-protein interactions needed to facilitate gene expression. In order to further characterize the tertiary complex between the beta - globin cis-acting elements with BP1, BP2, and HMG1+2 proteins, chimeric constructs were created. Placing proximal BP1 and BP2 binding sites in either a cis- or trans- position to each other (by inserting 1/2 or 1 full turn of DNA, respectively) did not markedly affect beta -globin silencing, suggesting the silencing function does not require specific proximal protein-protein interaction. However, inserting an arbitrary 22 bp fragment (creating two full turns of DNA) increased gene expression 3- fold compared to wt beta -expression levels, indicating proximal BP1 and BP2 spatial constraints required for optimal silencing. This negative distance constraint could be overcome by inserting a HMG1+2 DNA motif (which also correlates to the addition of two full turns of DNA) shown here to induce DNA flexure in the beta- globin gene, re-established BP1-BP2 silencing stabilization. Through the study of the combinatorial DNA cis-acting elements and trans-acting factors, a molecular mechanism for the developmental silencing of the human adult beta globin gene repression is evolving.